Cryptosporidium (Crypto), an intestinal parasite, causes diarrheal disease in humans worldwide. In the resource-constrained world, it is a leading cause of diarrhea and death in young children and a major opportunistic infection in untreated HIV/AIDS patients, in whom it can lead to severe, and ultimately fatal diarrhea and wasting. In the USA and other developed countries, it is a frequent cause of waterborne outbreaks of disease. Despite the global impact of Crypto, treatment options for cryptosporidiosis are severely limited. Nitazoxanide is the only FDA-approved drug for cryptosporidiosis. Thus, development of new interventions for cryptosporidiosis is imperative. There are several constraints to drug development for cryptosporidiosis. The pathogenesis of the disease and host-parasite interactions amenable to targeting for drug development are poorly understood. A major limitation to understanding pathogenesis is the lack of a primary human intestinal epithelial cell (IEC) model for infection and propagation of the parasite. Although cancer-derived or primary IEC lines grown in 2D culture support temporary Crypto infection the parasite cannot be propagated in these cells for more than 5 days. Genetic modification of Crypto has recently been pioneered but is impeded by the lack of an in vitro system to propagate transgenic parasites. Thus, a primary human intestinal model that supports infection and propagation of wild type and transgenic Crypto and recapitulates human gut structure and function is essential. We previously reported the construction and characterization of a bioengineered 3D human intestinal model in vitro. Our preliminary data indicate that this model can support Crypto infection for at least 17 days and that Crypto from infected scaffolds can re-infect new scaffolds for up to three cycles. However, IEC lines used in this model are cancer-derived and may not recapitulate normal human IEC structure and function. Enteroids are functional 3-D intestinal epithelial units derived from adult intestinal crypt stem cells and can be infected with Crypto. The overall goal of this project is to establish the bioengineered 3D model using human enteroids or primary human IEC lines for Crypto infection. Our central hypothesis is that this model will support infection and propagation of Crypto, expedite genetic manipulation of the parasite and testing of interventions for cryptosporidiosis. The Specific Aims are to 1) Establish a bioengineered 3D primary human intestinal tissue model and 2) Develop and optimize this model for infection and propagation of Crypto. Success in establishing this model and achieving propagation of infection in the model will be transformative for Crypto research. The model can be used to study the pathophysiology and host-parasite-microbe interactions in human cryptosporidiosis, expedite genetic manipulation of the parasite and test targeted, effective interventions for cryptosporidiosis.